Reduced Thiol Groups Research Articles

Interaction of Thiol Antioxidants with α,β-Unsaturated Ketone Moiety: Its Implication for Stability and Bioactivity of Curcuminoids

Many biological functions of curcumin have been reported. As certain bioactivities of curcumin are eliminated by antioxidants, reactive oxygen species generated by curcumin have been suggested as a relevant mechanism. In the present study, the effects of different types of antioxidants on the stability and bioactivities of curcumin were analyzed. High concentrations (>4 mM) of thiol antioxidants, including N-acetylcysteine (NAC), glutathione (GSH), and β-mercaptoethanol, accelerated the decomposition of curcumin and other curcuminoids; the submillimolar levels (<0.5 mM) of GSH and NAC rather improved their stability. Ascorbic acid or superoxide dismutase also stabilized curcumin, regardless of their concentration. The cellular levels and bioactivities of curcumin, including its cytotoxicity and the induction of heme oxygenase-1, were significantly reduced in the presence of 8 mM of GSH and NAC. The effects were enhanced in the presence of submillilmolar GSH and NAC, or non-thiol antioxidants. The present results indicate that antioxidants with a reduced thiol group could directly interact with the α,β-unsaturated carbonyl moiety of curcuminoids and modulate their stability and bioactivity.

The adaptive response to alternative carbon sources in the pathogen Candida albicans involves a remodeling of thiol- and glutathione-dependent redox status.

Candida albicans is an opportunist pathogen responsible for a large spectrum of infections, from superficial mycosis to systemic diseases known as candidiasis. During infection in vivo, Candida albicans must adapt to host microenvironments and this adaptive response is crucial for the survival of this organism, as it facilitates the effective assimilation of alternative carbon sources others than glucose. We performed a global proteomic analysis on the global changes in protein abundance in response to changes in micronutrient levels, and, in parallel, explored changes in the intracellular redox and metabolic status of the cells. We show here that each of the carbon sources considered - glucose, acetate and lactate - induces a unique pattern of response in C. albicans cells, and that some conditions trigger an original and specific adaptive response involving the adaptation of metabolic pathways, but also a complete remodeling of thiol-dependent antioxidant defenses. Protein S-thiolation and the overproduction of reduced glutathione are two components of the response to high glucose concentration. In the presence of acetate, glutathione-dependent oxidative stress occurs, reduced thiol groups bind to proteins, and glutathione is exported out of the cells, these changes probably being triggered by an increase in glutathione-S-transferases. Overall, our results suggest that the role of cellular redox status regulation and defenses against oxidative stress, including the thiol- and glutathione-dependent response, in the adaptive response of C. albicans to alternative carbon sources should be reconsidered.

Assessment of Oxidative Stress Biomarkers in Rat Blood.

Redox status assessments are time-consuming, require a large volume of samples and great reagent amounts, and are not adequately described for methodological reproducibility. Here, the objective was to standardize redox balance determination, based on previously described spectrophotometric tests in pregnant rats, to improve precision, time dispensed, and the volume of samples and reagents, while maintaining accuracy and adequate cost benefits. This protocol summarizes oxidative stress markers, which focus on spectrophotometric tests for the assessment of thiobarbituric acid-reactive substances, reduced thiol groups, and hydrogen peroxide, as well as the antioxidant activity of superoxide dismutase, glutathione peroxidase, and catalase in washed erythrocyte and serum samples from full-term pregnant rats. For non-pregnant rats and other species, it is necessary to standardize these determinations, especially the sample volume. All measurements were normalized by the estimated protein concentrations in each sample. To establish optimum conditions for the reproducibility of the proposed methods, we describe all changes made in each assay's steps based on the reference method reassessed for the new standardizations. Furthermore, the calculations of the concentrations or activities of each marker are presented. Thus, we demonstrate that the analysis of serum samples is easier and faster, but it is impossible to detect catalase activity. Furthermore, the proposed methods can be applied for redox balance determination, especially using smaller reagent amounts and lower sample volumes in lesser time without losing accuracy, as is required in obtaining samples during rat pregnancy.

Benefits of Vitamin D Supplementation on Pregnancy of Rats with Pregestational Diabetes and Their Offspring.

Studies on vitamin D supplementation have been performed in experimental and clinical investigations considering gestational diabetes and/or vitamin D deficiency in pregnancy. However, the results are controversial and few present the effects and mechanisms of this micronutrient on pregestational diabetes. The objective of this study was to evaluate the effect of vitamin D on the pregnancy of rats with pre-existing diabetes and their fetuses. Pregestational diabetes was induced in Sprague-Dawley rats at birth. The adult diabetic and nondiabetic rats were orally administered with vitamin D (cholecalciferol) throughout the pregnancy. The diabetes status was monitored during pregnancy by an oral glucose tolerance test (OGTT). At the end of the pregnancy, pancreas and blood samples were collected for morphological analyses and lipid peroxidation measurements, respectively. The influence of vitamin D treatment on reproductive outcomes, fetal growth, and development were compared to those of untreated diabetic and nondiabetic pregnant rats. P < 0.05 was considered a significant statistical limit. The diabetic rats given vitamin D had a greater number of insulin-positive cells, contributing to reduced blood glucose levels and thiobarbituric acid reactive substance concentrations (TBARS-an indicator of membrane lipid peroxidation), and increased reduced thiol group levels, contributing to suitable intrauterine conditions for better fetal development, which was confirmed by higher fetal viability rates. Thus, this study shows the effects and mechanisms of vitamin D supplementation on pre-existing diabetes in pregnant rats, confirming its beneficial effects on maternal redox status and glycemic control, and the decline of adverse maternal-fetal repercussions.

Recovery of reduced thiol groups by superoxide-mediated denitrosation of nitrosothiols

Nitrosation of critical thiols has been elaborated as reversible posttranslational modification with regulatory function in multiple disorders. Reversibility of S-nitrosation is generally associated with enzyme-mediated one-electron reductions, catalyzed by the thioredoxin system, or by nitrosoglutathione reductase.In the present study, we confirm previous evidence for a non-enzymatic de-nitrosation of nitrosoglutathione (GSNO) by superoxide. The interaction leads to the release of nitric oxide that subsequently interacts with a second molecule of superoxide (O2•−) to form peroxynitrite. Despite the formation of peroxynitrite, approximately 40–70% of GSNO yielded reduced glutathione (GSH), depending on the applied analytical assay. The concept of O2•− dependent denitrosation was then applied to S-nitrosated enzymes. S-nitrosation of isocitrate dehydrogenase (ICDH; NADP+-dependent) was accompanied by an inhibition of the enzyme and could be reversed by dithiothreitol. Treatment of nitrosated ICDH with O2•− indicated ca. 50% recovery of enzyme activity. Remaining inhibition was largely consequence of oxidative modifications evoked either by O2•− or by peroxynitrite. Recovery of activity in S-nitrosated enzymes by O2•− appears relevant only for selected examples. In contrast, recovery of reduced glutathione from the interaction of GSNO with O2•− could represent a mechanism to regain reducing equivalents in situations of excess O2•− formation, e.g. in the reperfusion phase after ischemia.

The Predictive Value of First-trimester Thiol/Disulfide Homeostasis in Preeclampsia.

To investigate the efficacy of first-trimester thiol/disulfide homeostasis (t/dh), a new oxidative stress marker, in predicting preeclampsia. Prospective cohort study. Department of Obstetrics and Gynecology, University of Health Sciences, Zeynep Kamil Women and Children Diseases Training and Research Hospital, Istanbul, Turkey, Department of Obstetrics and Gynecology, Bursa Yüksek Ihtisas Training and Research Hospital, Bursa, Turkey, from March 2016 to February 2019. In this multi-centre,serum samples of women with839 singleton pregnancies were collected between 11+0 to 13+6gestational weeks. A total of 215 singleton pregnant women were included in the study. The patient group consisted of 38 women, who were diagnosed with preeclampsia; while the control group consisted of 177 healthy pregnant women without any complication during pregnancy and after delivery. Totalthiol (TT) was estimated by the sum of existing thiol groups and reduced thiol groups (S-S and -SH). After the native thiols (-SH) and (TT) were determined, the disulfide (-SS) amounts, disulfide/total thiol percent ratios (-SS/-SH + -SS), disulfide/native thiol percent ratios (-SS/-SH), and native thiol/total thiol percent ratios (-SH/-SH + -SS) were calculated. There were no statistically significant differences between the groups in terms of[(-SH), (TT), (-SS), (-SS/-SH), (-SS/-SH + -SS), and (-SH/-SH + -SS)] six t/dh variables(p>0.05).The first-trimester body mass index (BMI) was statistically different between the two groups (p<0.001). In the receiver operating characteristic curve analysis, none of the concentrations of thiol levels and ratios was found to have a significant predictive value for preeclampsia. The BMI was a significant predictor for preeclampsia (area under curve: 0.749, p<0.001). Maternal serum t/dh at 11+0 to 13+6 weeks of gestation does not predict preeclampsia and t/dh may be the consequence rather than a cause in the pathogenesis of preeclampsia. Key Words: First-trimester, Preeclampsia, Sulfhydrylcompounds, Thiols.

Sodium iodate induces ferroptosis in human retinal pigment epithelium ARPE-19 cells

Sodium iodate (SI) is a widely used oxidant for generating retinal degeneration models by inducing the death of retinal pigment epithelium (RPE) cells. However, the mechanism of RPE cell death induced by SI remains unclear. In this study, we investigated the necrotic features of cultured human retinal pigment epithelium (ARPE-19) cells treated with SI and found that apoptosis or necroptosis was not the major death pathway. Instead, the death process was accompanied by significant elevation of intracellular labile iron level, ROS, and lipid peroxides which recapitulated the key features of ferroptosis. Ferroptosis inhibitors deferoxamine mesylate (DFO) and ferrostatin-1(Fer-1) partially prevented SI-induced cell death. Further studies revealed that SI treatment did not alter GPX4 (glutathione peroxidase 4) expression, but led to the depletion of reduced thiol groups, mainly intracellular GSH (reduced glutathione) and cysteine. The study on iron trafficking demonstrated that iron influx was not altered by SI treatment but iron efflux increased, indicating that the increase in labile iron was likely due to the release of sequestered iron. This hypothesis was verified by showing that SI directly promoted the release of labile iron from a cell-free lysate. We propose that SI depletes GSH, increases ROS, releases labile iron, and boosts lipid damage, which in turn results in ferroptosis in ARPE-19 cells.

Comparison of oxidative stress markers between single and twin gestations in Dorper ewes during pregnancy, delivery and postpartum

Abstract Pregnancy in sheep is a physiological condition characterized by a significant increase in energy demand to ensure adequate foetal growth and development, making them susceptible to oxidative stress. The aim of this study was to analyse the markers of oxidative stress in sheep during pregnancy, parturition and close to peripartum (up to 48 h postpartum), comparing single with twin gestation. Sixty healthy Dorper ewes from 2 to 5 years’ old raised under semi-intensive management were used in this study. Ewes were grouped based on foetal number: Group 1, with single pregnancy (n = 30); Group 2, with a twin pregnancy (n = 30). Blood samples were collected nine times: after artificial insemination (D0, control); at 30 days (D30), 90 days (D90), 120 days (D120), 130 days (D130), and 140 days (D140) of pregnancy; on the day of delivery (DD); at 24 h (PD1) and 48 h (PD2) post-delivery. The concentrations of reduced thiol groups (-SH, P = 0.0155), the enzymatic activities of superoxide dismutase (SOD, P = 0.0009), glutathione peroxidase (GSH-Px, P = 0.0110) and catalase (CAT, P = 0.0001) differed according to fetal number by time. Twin gestation decreased concentration of thiobarbituric acid reactive substances (TBARS; P = 0.0278), and TBARS had differed over time (P

Oxidative stress biomarkers in newborn calves: Comparison among artificial insemination, in vitro fertilization and cloning

Oxidative stress occurs when there is greater than optimal production of reactive oxygen species (ROS) or an antioxidant system failure. Calves produced using in vitro fertilization (IVF) or cloning (CA) have greater mortality rates, with greater incidence of respiratory diseases, which could be explained by the deleterious outcomes from oxidative stress. Calves were studied that were produced using: artificial insemination (AI; n = 20), in vitro fertilization (IVF; n = 15) or cloning (CA; n = 15). Blood samples were collected at 6, 12, 24 and 48 h subsequent to the time of birth. The cloned calves had greater ROS production from lipid peroxidation, with greater thiobarbituric acid reactive substances. This factor was associated with a lesser amount of superoxide dismutase in the CA. Calves produced using IVF had a greater activity of catalase and glutathione peroxidase, either due to greater production of hydrogen peroxide or greater efficiency of enzymatic response of these neonates. Calves produced using AI had greater concentrations ​​of reduced thiol groups. These associated factors may indicate there is greater oxidative stress in calves produced by IVF and cloning than with use of AI, however in these calves there was an effective response to these oxidative stressors within 48 h subsequent to birth. Hence, calves produced using IVF and by cloning have greater ROS production when compared to calves produced using AI. The calves produced using IVF, however, had a greater enzymatic activity or were more efficient in adapting to ROS when compared to calves produced by cloning.

Characterization of a Sulfhydryl Oxidase From Plasmodium berghei as a Target for Blocking Parasite Transmission.

Quiescin sulfhydryl oxidase (QSOX), present in a wide variety of eukaryotic species, catalyzes the insertion of disulfide bonds into unfolded, reduced proteins. Here we characterized the QSOX protein from the rodent malaria parasite Plasmodium berghei (PbQSOX), which is conserved in all sequenced malaria parasite species. The PbQSOX protein was not expressed in asexual erythrocytic stages, but was most abundantly expressed in ookinetes. Indirect immunofluorescence assays revealed PbQSOX was not only localized in cytoplasm of gametocytes, gametes and ookinetes, but also expressed on the surface of gametes and ookinetes. Western blot identified extracellular presence of PbQSOX in the culture medium of ookinetes suggestive of secretion. Pbqsox deletion (Δpbqsox) did not affect asexual intraerythrocytic development, but reduced exflagellation of male gametocytes as well as formation and maturation of ookinetes. Pbqsox deletion also led to a significant increase in the reduced thiol groups of ookinete surface proteins, suggesting that it may play a role in maintaining the integrity of disulfide bonds of surface proteins, which might be needed for ookinete development. Mosquitoes that fed on Δpbqsox-infected mice showed a significant reduction in ookinete and oocyst numbers compared to those fed on wild-type parasite-infected mice. Further, both polyclonal mouse antisera and a monoclonal antibody against the recombinant PbQSOX exhibited substantial transmission-blocking activities in in vitro and mosquito feeding assays, suggesting QSOX is a potential target for blocking parasite transmission.

Molecular mechanisms of the nephroprotective action of polyphenols in experimental chronic kidney disease. Association with hydrogen sulfide system

Annotation. The results of a study of the effects of genistein, resveratrol and quercetin on the main markers of oxidative stress, thiol disulfide equilibrium and the state of the nitric oxide system in rat kidneys after modelling chronic kidney disease (CKD) (5/6 of a single kidney nephrectomy) is shown. Blood and single rat kidney homogenate were used for biochemical studies. Statistical processing of the obtained results was performed in the program “STATISTICA 6.1”. The results are presented as M±m. The significance of the difference between the scores was evaluated using the Student's t-test or the Mann-Whitney U-test. A nonparametric Spearman correlation coefficient was used to determine the correlation between two independent indicators. It was shown that the studied substances, especially quercetin, caused antioxidant activity in CKD, which was manifested by a significant decrease of MDA, CGP and NADPH oxidase activity (by 21.0–40.4%), as well as an increase in SOD activity (by 29.7–38.7%) in kidney compared with untreated animals. Reliable strong correlation (r=ǀ0.65–0.74ǀ) between the H2S content and oxidative stress markers was established. The use of polyphenols in CKD reduced the thiol-disulfide imbalance in rat kidneys: the activity of thioredoxin reductase significantly increased, the content of reduced thiol groups of proteins (by 16.0–66.4%) and the level of disulfides decreased (by 21.1–25.3%), in comparison with untreated animals. Stabilization of the thiol disulfide equilibrium is one of the mechanisms of the effect of polyphenols on the H2S system in the kidneys, since significant and strong correlation relationships were recorded between these indicators and the H2S content (r=ǀ0.63–0.75ǀ). The administration of resveratrol, and especially genistein, was accompanied by an increase in the activity of the eNOS (by 36.3–44.7%) and a decrease in the activity of iNOS (by 15.4–20.9%), in comparison with untreated animals. The NO system in the kidneys is an important molecular target for influence of genistein and resveratrol on the production of H2S during CKD is realized, since reliable strong correlation were recorded between the H2S content and the activity of NO synthase isoforms (r=ǀ0.70–0.75ǀ).

An enhanced fluorescence sensor for specific detection Cys over Hcy/GSH and its bioimaging in living cells.

Cysteine (Cys) is not only the central matter of sulfur metabolism in cells but also the only amino acid with reduced thiol group in 20 kinds of natural amino acids. In animal cells, Cys is taking part in many important and essential biological functions including protein synthesis, detoxification and metabolism. The development and application of fluorescent probes for the detection of Cys have attracted more and more attention and interest. Herein, we report a new fluorescent probe NFA that utilized naphthyl carboxy fluorescein as fluorophore and acryloyl group as reaction site for Cys specific detection. The probe essentially has weak fluorescence. Cys addition to NFA containing system induced distinct enhanced fluorescence emission which was attributed to the nucleophilic reaction of cysteine and acryloyl to release the fluorophore. The signal fluorescent response detection system allows NFA to be a reliable tool for Cys detection with low detection limit (0.58 μM). And NFA has been successfully applied for Cys imaging specifically in live Hela cells, which promotes the probe as a potential tool to understand the pathology of Cys related diseases.

Primary targets of the sesquiterpene lactone deoxymikanolide on Trypanosoma cruzi

BackgroundDeoxymikanolide is a sesquiterpene lactone isolated from Mikania micrantha and M. variifolia which, has previously demonstrated in vitro activity on Trypanosoma cruzi and in vivo activity on an infected mouse model. PurposeBased on these promising findings, the aim of this study was to investigate the mechanism of action of this compound on different parasite targets. MethodsThe interaction of deoxymikanolide with hemin was examined under reducing and non- reducing conditions by measuring modifications in the Soret absorption band of hemin; the thiol interaction was determined spectrophotometrically through its reaction with 5,5′-dithiobis-2-nitrobenzoate in the presence of glutathione; activity on the parasite antioxidant system was evaluated by measuring the activity of the superoxide dismutase and trypanothione reductase enzymes, together with the intracellular oxidative state by flow cytometry. Superoxide dismutase and trypanothione reductase activities were spectrophotometrically tested. Cell viability, phosphatidylserine exposure and mitochondrial membrane potential were assessed by means of propidium iodide, annexin-V and rhodamine 123 staining, respectively; sterols were qualitatively and quantitatively tested by TLC; ultrastructural changes were analyzed by transmission electron microscopy. Autophagic cells were detected by staining with monodansylcadaverine. ResultsDeoxymikanolide decreased the number of reduced thiol groups within the parasites, which led to their subsequent vulnerability to oxidative stress. Treatment of the parasites with the compound produced a depolarization of the mitochondrial membrane even though the plasma membrane permeabilization was not affected. Deoxymikanolide did not affect the intracellular redox state and so the mitochondrial dysfunction produced by this compound could not be attributed to ROS generation. The antioxidant defense system was affected by deoxymikanolide at twenty four hours of treatment, when both an increased oxidative stress and decreased activity of superoxide dismutase and trypanothione reductase (40 and 60% respectively) were observed. Both the oxidative stress and mitochondrial dysfunction induce parasite death by apoptosis and autophagy. ConclusionBased on our results, deoxymikanolide would exert its anti-T cruzi activity as a strong thiol blocking agent and by producing mitochondrial dysfunction.

Interaction of metals from group 10 (Ni, Pd, and Pt) and 11 (Cu, Ag, and Au) with human blood δ-ALA-D: in vitro and in silico studies.

Mammalian δ-aminolevulinate dehydratase (δ-ALA-D) is a metalloenzyme, which requires Zn(II) and reduced thiol groups for catalytic activity, and is an important molecular target for the widespread environmental toxic metals. The δ-ALA-D inhibition mechanism by metals of Group 10 (Ni, Pd, and Pt) and 11 (Cu, Ag, and Au) of the periodic table has not yet been determined. The objective of this study was to characterize the molecular mechanism of δ-ALA-D inhibition caused by the elements of groups 10 and 11 using in vitro (δ-ALA-D activity from human erythrocytes) and in silico (docking simulations) methods. Our results showed that Ni(II) and Pd(II) caused a small inhibition (~ 10%) of the δ-ALA-D. Pt(II) and Pt(IV) significantly inhibited the enzyme (75% and 44%, respectively), but this inhibition was attenuated by Zn(II) and dithiothreitol (DTT). In group 11, all metals inhibited δ-ALA-D with great potency (~ 70-90%). In the presence of Zn(II) and DTT, the enzyme activity was restored to the control levels. The in silico molecular docking data suggest that the coordination of the ions Pt(II), Pt(IV), Cu(II), Ag(I), and Au(III) with thiolates groups from C135 and C143 residues from the δ-ALA-D active site are crucial to the enzyme inhibition. The results indicate that a possible mechanism of inhibition of δ-ALA-D by these metals may involve the replacement of the Zn(II) from the active site and/or the cysteinyl residue oxidation.

A new molecular switch based on a symmetrical dinuclear complex of two tricarbonylrhenium(I) moieties bridged by 4,4″-azobis-(2,2′-bipyridine)

A new symmetrical dinuclear complex of two tricarbonylrhenium(I) moieties, of formula [(CH3CN)(CO)3Re(4,4″-azobpy) Re(CO)3(CH3CN)](PF6)2, with 4,4″-azobpy = 4,4″-azobis-(2,2′-bipyridine), has been synthesized and characterized by spectroscopic, electrochemical, spectroelectrochemical, photophysical and computational techniques. The bridging azo group in the bipyridyl ring decreases the emission quantum yield of the 3MLCT lowest-lying excited state respect to similar Re(I) complexes and introduces a new emissive excited state with a longer lifetime, due to increased electronic delocalization in the bridging ligand. When reducing the azo group in CH3CN/H2O mixtures with sodium dithionite, the emission is enhanced by an order of magnitude. Therefore, this complex can be used as a “molecular switch” with electron and proton additions. Besides, changes in the absorption spectrum on addition of l-Cysteine can be applied for sensing aminoacids with reducing thiol groups. The electronic structures calculated by DFT methods agree reasonably well with experimental results.

Effects of heat stress and probiotic supplementation on protein functionality and oxidative stability of ground chicken leg meat during display storage.

The present study aimed to evaluate the effects of heat stress and probiotic supplementation on protein functionality and oxidative stability of ground chicken leg during display storage. Two hundred and forty, 1-day-old male chicks (5 birds per pen) were subjected to four treatments in a 2 (thermoneutral condition at 21 °C and cyclic heat stress at 32-21-32 °C for 10 h day-1 ) × 2 (regular diet with 0 or 0.25 g kg-1 Bacillus subtilis) factorial design. Chickens were harvested at day 46, and pairs of whole legs were collected at 1 day postmortem. The chicken legs were deboned, ground, tray-packaged with oxygen-permeable film, and displayed for 3 days. Heat stress and probiotic supplementation had no impact on pH, water-holding capacity, color, protein functionality, lipid lipolysis and lipid/protein oxidation stability (P > 0.05). Display storage increased the pH and lipid oxidation of ground chicken legs (P < 0.05). In addition, protein oxidation occurred during display storage, as determined via an increased carbonyl group (P = 0.0109) and reduced thiol group (P < 0.0001). The results of the present study indicate that chronic heat stress and probiotic supplementation had no practical adverse impact on protein functionality and oxidative stability of ground chicken leg meat. © 2017 Society of Chemical Industry.

The Application of Clove Extract Protects Chinese-style Sausages against Oxidation and Quality Deterioration

This study was conducted to evaluate the effects of clove extract (CE) (0.25%, 0.5%, 1%, and 2%) on the oxidative stability and quality deterioration of Chinese-style sausage stored for 21 d at 4°C. The addition of clove extract to sausages significantly retarded increases in Thiobarbituric Reactive Substances (TBARS) values (p<0.05), while also controlling the production of protein carbonyls (p<0.05). However, the addition of clove extract promoted reduced thiol group content in sausages (p<0.05). Sausages amended with clove extract also had decreased L* values (p<0.05) and increased a* values (p<0.05) when compared with the control. Similarly, texture deterioration was retarded in sausage containing added clove extract when compared with the control during refrigerated storage. Moreover, the addition of clove extract had no negative effects on the sensory properties of sausages. These results suggested that clove extract was effective at protecting sausages from oxidation and quality deterioration during refrigerated storage for 21 d.

COMPARATIVE ANALYSIS OF THE DYNAMICS OF IMMUNE-ANTIOXIDANT STATUS OF THE ANTIOXIDANT SYSTEM IN PATIENTS SUFFERING FROM HYPOTHYROIDISM AND COPD

Aim . To carry out a comparative study of the features and shift tendency of individual indicators of the antioxidant system in patients with hypothyroidism and COPD of various phenotypes. 79 patients suffering from COPD III and IV were examined at the age of 60 to 80 years (men and women) who were hospitalized in the pulmonology department of the Regional Clinical Hospital No. 2 in Krasnodar, along with 47 patients suffering from hypothyroidism. Materials and methods. The state of the blood antioxidant system was assessed by the activity of erythrocyte catalase and superoxide dismutase, the level of reduced thiol groups of erythrocytes, and the amperometric index of the total antioxidant activity of blood plasma. The activity of catalase was studied in the hemolysate of erythrocytes according to the method of Beers R. Results. It was established that there were significant changes in the functioning of individual links of the antioxidant protection system in patients with COPD and hypothyroidism, as well as the stress in the system in general. At the same time, enzyme activity of the antioxidant system of erythrocytes varies in multidirectional ways, whereas the indices of the cellular and extracellular non-enzyme links of the endogenous antioxidant system change in a unidirectional way towards the decrease, which may indicate a significant activation of free radical oxidation processes and partial depletion of the low-molecular antioxidant blood link. Conclusion. The revealed imbalance in the pro-/antioxidant system requires the introduction of drugs with antioxidant activity into therapeutic regimens. In patients with COPD, hypoxia and chronic inflammation may provoke oxidative stress, while in patients with hypothyroidism –decrease in the level of basal metabolism and anabolic processes in general.

312 - The Mechanism of MnTE-2-PyP Functioning Differently in Cancer Cells and Normal Cells

Rationale: Radiotherapy reduces prostate cancer growth but patients suffer from side effects caused by the accumulation of reactive oxygen species. MnTE-2-PyP5+ reduces radiation induced side effects, while simultaneously inhibiting cancer progression. We hypothesize that MnTE-2-PyP increases H2O2 levels in cancer cells but not normal cells due to the difference in redox capacities between the two cell types. Methods Three cell lines, PC3, LNCaP, primary mouse fibroblasts(PMF) were treated with PBS or 30µM MnTE-2-PyP. A Ros-Glo assay was performed to measure H2O2 levels in MnTE-2- PyP treated or untreated cells. In order to measure protein oxidation in PC3 cells treated with MnTE-2-PyP, biotinylated iodoacetamide (BIAM) probe was used which binds only to reduced thiol groups. A western for streptavidin was performed to identify proteins containing reduced thiols. Mass Spec was performed to determine protein contents difference in both drug treated or untreated cells. An in gel catalase activity assay was performed for cells treated with or without MnTE-2-PyP. The EPR assay was conducted to detect the basal levels of O2– in 3 cell lines. Results MnTE-2-PyP significantly increased H2O2 level in PC3 cells and LNCaP cells as compared to PBS treated cells but no in normal primary fibroblasts. Accordingly, we found that the increased H2O2 in the cancer cells caused global oxidation of thiols in many different proteins. MnTE-2-PyP treatment increased the amount of oxidized proteins in PC3 cells but not in PMF. Mass spectrometry was performed to identify oxidized proteins in the presence of MnTE-2-PyP. Preliminary results have identified two proteins that are oxidized with MnTE-2-PyP treatment: serine/threonine-protein phosphatase PP1-beta catalytic subunit and Pre-mRNA-processing factor 19. One explanation for why more H2O2 is produced in cancer cells vs. normal cells is that normal cells may have more active H2O2 removing enzymes than cancer cells, like catalase. However, we found that LNCaP cells have the highest catalase activity than PMF and PC3. But MnTE-2-PyP did not change the catalase activity in any kind of cell line. We also measured basal levels of superoxide production in cancer vs. normal cells, since MnTE-2-PyP scavenges superoxide to produce H2O2. We found that PMF have higher basal superoxide radicals than PC3 cells and LNCaP cells. Conclusion MnTE-2-PyP increases H2O2 levels in cancer cells but not normal cells, which leads to the oxidation of proteins in PC3 cells. We hypothesized that this difference in H2O2 production could be due to the fact that cancer cells have a reduced ability to remove H2O2; however, our preliminary data do not support this hypothesis. There are other important H2O2 removing enzymes that we still need to measure to obtain the over antioxidant capacity of the cells.

Effects of Freeze-Thaw and Storage on Enzymatic Activities, Protein Oxidative Damage, and Immunocontent of the Blood, Liver, and Brain of Rats.

Most scientific studies are too long to be conducted in a single day or even in a few days. Thus, there is a need to store samples for subsequent investigations. There is sparse information about specific sample storage protocols that minimize analytical error and variability in evaluations of redox parameters. Therefore, the effects of storage temperature and freezing time on enzymatic activities, protein oxidative damage, and CAT (catalase) and SOD1 (superoxide dismutase) immunocontent of blood, liver, and brain from rats were determined for two different sample forms (frozen homogenized tissue or frozen intact tissue). Superoxide dismutase activity was drastically decreased in blood and liver with an increase in freezing time, but not in brain. Catalase activity showed a decrease only in intact liver at -20 and -80°C. In contrast, in blood it showed an increase in intact tissue at -20 and -80°C. Reduced thiol groups generally decreased with freezing time, but showed an increase in intact blood at -20 and -80°C, probably because of color interference. Carbonyl groups in homogenized liver and brain, and in intact blood (except at 80°C) drastically increased with freezing time. Freezing time did not modulate the immunocontent of CAT and SOD1 levels in any tissue. In conclusion, our results indicate that storage at -20°C affects redox parameters more than storage at -80°C. Storage for a long time may compromise the samples, leading to changing parameters due to oxidative stress. Thus, we suggest processing the samples as soon as possible. However, if this is not possible, then material can be aliquoted into different tubes to prevent the effect of refreezing of samples.